Diseases of iron metabolism continue to be a major health concern in the United States. Iron deficiency remains the most common single nutrient deficiency in the US and individuals experience negative health consequences due to iron deficiency in the absence of anemia, primarily due to alterations in iron metabolism in skeletal muscle. Surprisingly little is known about the molecular mechanisms regulating iron metabolism in skeletal muscle and how alterations in this tissue affects iron homeostasis. The long-term research goal of our laboratory is to advance understanding of how iron metabolism coordinated, and how alterations in iron sensing can lead to the development or prevention of disease. There is evidence suggesting expression of genes involved in iron metabolism is regulated by ID, however, the molecular mechanisms remain poorly characterized. Only recently has the role of small regulatory RNA molecules called microRNAs (miRNAs) been identified as an important mechanism for regulating various cellular processes. Our preliminary in silico analysis of miRNA targets resulted in the identification of mRNAs encoding proteins involved in iron metabolism. Thus our primary objectives are to determine the extent to which expression of miRNAs is regulated in response to ID and to characterize the impact of miRNA expression on potential regulatory targets involved in iron metabolism. We will critically evaluate targets of differentially expressed miRNAs and examine the roles of these targets in cellular iron metabolism. The central hypothesis is that miRNA expression is regulated in response to ID and that changes in expression are associated with changes in the expression target mRNAs resulting in the homeostatic regulation of cellular iron metabolism. To test this hypothesis, this proposal encompasses two specific aims. In Aim 1 we will evaluate and characterize iron-dependent changes in miRNA expression using a weanling rat model of ID. In Aim 2 we will determine the extent to which miRNAs contribute to the regulation cellular iron metabolism through modulating the expression of proteins involved in cellular iron homeostasis. MicroRNA expression profiles in the liver and skeletal muscle will be determined using miRNA microarrays and validated using qPCR. Combining in vivo and in vitro studies, we will identify and characterize target mRNAs and determine the role of these mRNAs in iron metabolism. We will also determine the extent to which changes in miRNA expression are due to alterations in iron status versus alterations in iron sensing. The proposed research is relevant to the maintenance of optimal health in light of the prevalence ID, and by taking an interdisciplinary approach to examine the relationships between iron status and cellular metabolism, we will expand our understanding of how iron contributes to the maintenance of optimal health. PUBLIC HEALTH RELEVANCE: Iron deficiency continues to be the most common nutrient deficiency and is associated with alterations ranging from impairment in immune function, delayed cognitive development, and decreased capacity of work in skeletal muscle. The focus of this project is to examine the extent to which iron status is associated with alterations in microRNA expression that may contribute to some of the tissue-specific effects of iron deficiency. The results of this study will inform our understanding of the molecular mechanisms coordinating the cellular response to iron deficiency and may have implications in understanding alterations in iron metabolism observed in chronic diseases such as cancer and diabetes.